CD8+ T cells are critical components of the anti-viral immune response to some viruses. Naive CD8+ T cells differentiate into effector cells only after recognition of peptide-MHC Class I complexes on the surface of antigen presenting cells (APC). The peptides presented can be generated from protein antigens via a number of pathways in vivo. Peptides can be produced from antigens expressed within a virus-infected APC, when presentation is termed direct-priming. Alternatively, peptides can be produced from proteins that are transferred from other cells to APC prior to presentation, a process known as cross-priming. The extent to which direct-priming or cross-priming contribute to activation of naive CD8+ T cells in vivo is not known. The overall objective of this project is to delineate the mechanisms used to generate MHC Class I-peptide complexes during priming of naive CD8+ T cells in vivo. Our underlying hypothesis is that alteration in protein expression can, and does, direct in vivo antigen presentation into direct or cross-priming pathways. To examine this issue we will use recombinant viruses to express multiple protein antigens and will analyze antigen presentation to naive and effector CD8+ T cells both in vitro and in vivo. ln Aim 1 we will determine the effects of targeting viral antigen for expression in specific tissues on the pathways of antigen presentation used in vivo. We will examine the timing and efficiency of CD8+ T cell priming to ubiquitously expressed or tissue-targeted antigen. We will also identify the APC responsible for priming via each route, and examine the properties of this APC that are necessary for effective priming. In Aim 2 we will examine differential antigen presentation as a mechanism for the reduced immunogenicity of virus genes expressed late in the virus life cycle. In addition, by using a natural example of antigen shuttled into different antigen presentation pathways in vivo we will compare the efficiency of CD8+ T cell priming via direct or cross-priming to the amount of antigen made in vivo. In Aim 3 we will determine, in vitro and in vivo, the characteristics of proteins that are preferentially transferred from virus-infected cells to APC during cross-priming. Delineation of the mechanisms governing the use of different antigen presentation pathways in vivo will provide a basis for the rational design of vaccines and immunotherapeutic strategies aimed at induction of protective CD8+ T cells.